Project summary. The native microbiota of mosquitoes profoundly influences many aspects of mosquito biology, including the ability of mosquitoes to transmit arboviral pathogens. Perturbation of the mosquito microbiome, either by supplementation of antibiotics or by oral inoculation of bacterial isolates into the mosquito gut can interfere with flaviruses such as Dengue virus (DENV) and West Nile Virus as well as alphaviruses like Chikungyunya (CHIKV). Most studies indicate that gut microbes are antagonistic to pathogens, but particular bacteria facilitate viral infection in mosquitoes. Additionally, we are beginning to appreciate that arbovirus infection in mosquitoes can alter the bacterial microbiome. Taken together, it is clear that there is complex interplay, either by direct or indirect mechanisms, between native gut microbes of mosquitoes and invading arbovirus, and these interactions have a profound effect on vector competence. Here we will characterize the interplay between gut microbes of Aedes mosquitoes and Zika virus to determine how these interactions influence ZIKV vector competence in mosquitoes. Our work will focus on both bacterial and fungal taxa of Aedes aegypti and Aedes albopictus. While it is emerging that bacterial symbionts alter vector competence, we have a poor understanding of the role of fungal microbes on mosquito biology and vector competence. Our work will exploit high throughput sequencing technologies to obtain a culture-independent quantitative characterization of the bacterial and fungal microbiome. Importantly, experiments will use both lab-reared and field-collected mosquitoes as our appreciation of the role of the microbiome of on vector competence of mosquitoes in the field is poorly understood. In specific aim 1, we will identify bacterial and fungal taxa that alter vector competence by high throughput sequencing comparing perturbed and conventionally reared mosquitoes. Using field-collected mosquitoes, ZIKV vector competence will be correlated to microbial abundance and composition. As the composition of the microbiome is variable, the presence or abundance of specific symbionts may account for variation in arbovirus vector competence. ZIKV vector competence assays will be completed on gnotobiotic mosquito lines that have had isolates reinfected, functionally validating candidate taxa identified from our sequencing experiments. In specific aim 2, we will examine how ZIKV infection alters the bacterial and fungal microbiome using high throughput sequence. We will determine if microbes that ZIKV inhibits have anti-viral properties and examine if ZIKV-mediated modulation of the microbiome is transferred between generations. Lastly, we will determine if such transgeneration alteration of the micobiome affects vector competence of subsequent generations to ZIKV. This work will provide insights in microbial interactions within mosquito, a poorly understudied but important area of vector biology that has promising applications to control ZIKV and other mosquito-borne viral disease. This project will lay the foundation for future work in the Hughes laboratory, which is to develop applied novel microbial-based strategies suitable for simultaneously controlling multiple arboviruses such as ZIKV, DENV, CHIKV, and yellow fever virus.